The reasons organisms encode multiple Hsp70s are that it provides broader capacity to regulate abundance of Hsp70 in accordance with needs, or that having different Hsp70s provides similar, but distinct functions important for carrying out specific tasks either within cells in general or in specific types of cells. Earlier we constructed a yeast system to evaluate functions of Hsp70 from any source. We are using it to investigate how Hsp70s from both within and across species perform with regard to providing essential activities for cell growth and for propagation of different prions. This very sensitive system gives us the unique ability to distinguish exquisite functional differences among nearly identical Hsp70 isoforms and provides a means to approach the problem of uncovering the underlying mechanisms. Previously we showed the four S. cerevisiae Hsp70s (Ssa1, Ssa2, Ssa3 and Ssa4) possess distinct functions as revealed by the different ways they affect the propagation of different prions. These differences in the ways the prions depend on subtle distinctions in Hsp70 activities provide a sensitive way to monitor functional distinctions among the Hsp70s. For example, we identified a specific residue in the ATPase domain of the 98% identical Ssa1 and Ssa2 (A83 or G83, respectively) as solely responsible for a functional distinction between Ssa1p and Ssa2p that is critical for a vacuolar import protein degradation pathway. Our findings show that exquisitely fine differences in structure can underlie distinctions in function of nearly identical Hsp70s and that regulation of Hsp70 substrate interactions, rather than substrate binding per se, is the important parameter that determines functional distinctions. In the cell these subtle differences can lead to large-scale effects on cellular processes and might be all that is needed to direct different Hsp70s to specialized tasks. Our continued work focuses on determining if differences in functions of various Hsp70s are mediated by differences in the way they cooperate with other components of the cellular protein quality control machinery, such as co-chaperones or other major chaperones. We are also interested in learning whether such differences in Hsp70 function contribute to protection from amyloid toxicity that we see in some of our strains, and the extent to which human Hsp70s might possess such protective functions. Elevating Hsp70 can moderate pathology in models of protein folding disorders, while in the same models reducing Hsp70 activity can exacerbate, or alone even cause, pathology. Hsp70 is therefore a promising therapeutic candidate for amyloid and other protein folding disorders and it is being studied intensively as a drug target. Our work can help guide decisions about which Hsp70-family members would be most useful for such applications, or identify potential problems that could arise due to distinct sensitivities of different Hsp70s to specific compounds.